Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells

Abstract

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)] 2+(Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cellcycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMRdeficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.cancer | DNA damage response | metalloinsertor | mismatch repair D NA mismatch repair (MMR) corrects mismatched base pairs that arise from replication errors and is involved in the recognition and processing of DNA damage induced by chemotherapeutics such as cis-diamminedichloroplatinum(II) (cisplatin) (1, 2). Loss of function of the MMR pathway is an important mechanism for mutation generation and predisposition to cancer. Lynch syndrome, an inherited condition characterized by susceptibility to develop colon cancer, results from germline inactivation of the MMR pathway (3). Somatic loss of function of the MMR pathway occurs in 10-25% of cancers of the colon, endometrium, ovary, and other tissue types (3-5). MMR-deficient cancer cells display a 100-to 1,000-fold increase in mutation rate and are resistant to many DNA-targeted chemotherapeutics (6, 7). Because of this, patients with MMR-deficient cancers do not benefit from standard of care therapy including cisplatin (8, 9). New therapies that target these patient populations are needed.We have previously described a class of octahedral rhodium(III) complexes, termed metalloinsertors, as a strategy toward the targeted therapy of MMR-deficient cancers. These compounds contain a sterically expansive 5,6-chrysenequinone diimine ligand (chrysi; Fig. 1A) that binds with high specificity to DNA mismatches, independent of sequence context (10). X-ray crystal structures demonstrate that the chrysi ligand is capable of inserting at the mismatch from the DNA minor groove; this binding mode, called metalloinsertion, maintains duplex stacking but displaces the mismatched bases into the major or minor groove (11, 12). As a consequence, metalloinsertor binding preferentially inhibits DNA synthesis and growth of cell lines that are MMR-deficient (13-15). Structureactivity relationship studies reveal that compound selectivity and cell potency...

Cellular Target of a Rhodium Metalloinsertor is the DNA Base Pair Mismatch

“…We performed fluorescence titrations with RhCy3 and increasing amounts of gDNA extracted from at est set of eight cell lines that span deficiencies in different MMR genes (Table S2). [26] As can be seen in Figure 4, ac orrelation (r = À0.52) was observed between increasing RhCy3 fluorescence and decreasing IC50 of RhPPO. By removing the potential outlier,D U145 (the only cell line tested mutated in two MMR proteins), the correlation improves dramatically,( r = À0.81, p < 0.05), suggesting other factors may influence the cytotoxicity of RhPPO or fluorescenceo fRhCy3 in DU145.…”

“…Here we use this probe to better understand the cytotoxic effect of RhPPO on a panel of cancer cell lines, but these studies also demonstrate the powerful detection and diagnostics properties of RhCy3 in MMR− cancers. We performed fluorescence titrations with RhCy3 and increasing amounts of gDNA extracted from a test set of eight cell lines that span deficiencies in different MMR genes (Table S2) . As can be seen in Figure , a correlation ( r =−0.52) was observed between increasing RhCy3 fluorescence and decreasing IC50 of RhPPO .…”

A Family of Rhodium Complexes with Selective Toxicity toward Mismatch Repair-Deficient Cancers

“…19,29 In this structure-activity relationship study, we used ELISA and MTT assays to determine the effect of ligand substitution on biological activity in MMR-deficient and -proficient cells. The ELISA was used to determine the inhibitory effects on DNA synthesis and the MTT assay was performed to establish levels of cytotoxicity.…”

Cell-Selective Cytotoxicity of a Fluorescent Rhodium Metalloinsertor Conjugate Results from Irreversible DNA Damage at Base Pair Mismatches

“…[Rh(phen)(chrysi)(PPO)] 2+ (RhPPO) demonstrates remarkably high cellular potency and selectivity towards MMR-deficient cells relative to the first generation of compounds. 22,23 These findings were extended to a panel of 27 CRC cell lines, where…”

“…These results support the hypothesis that the DNA lesions induced by RhPPO-Cy3 binding to DNA mismatches are not able to be repaired in the presence of the compound and also prevent cross-talk between DNA checkpoint proteins and p53, the key mediator of the apoptotic pathway.DISCUSSIONDNA mismatches present in MMR-deficient cancer cells present an opportunity for selective therapeutic targeting. The rhodium metalloinsertor RhPPO is characterized by high cellular potency across MMR-deficient cell lines,22,24 and we have proposed that its buckled orientation in the DNA minor groove leads to a pronounced extrusion of the mismatched base pair from the DNA helix that activates DNA-binding proteins to generate DNA lesions 22,23. Crystallography efforts are ongoing to address the molecular structure of RhPPO bound to the DNA mismatch.…”